Uses of mammalian cytokines and agonists; related reagents

ABSTRACT

Provided are methods of treatment for tumors. In particular, provided are methods of using of a cytokine molecule and its receptor.

This application claims benefit of U.S. Provisional patent applicationSer. No. 60/488,263 filed Jul. 18, 2003.

FIELD OF THE INVENTION

The present invention relates generally to uses of mammalian cytokinemolecules and related reagents. More specifically, the invention relatesto identification of mammalian cytokine-like proteins and inhibitorsthereof that can be used in the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer is a progressive disease, occurring in a series of well-definedsteps, usually as a consequence of activating or deactivating mutations.These mutations often render proliferating cells self-sufficient forgrowth, insensitive to growth-inhibitory signals, resistant to programsof terminal differentiation, senescence, or apoptosis, as well asendowing them with unlimited self-renewal capacity, the ability toorchestrate and direct sustained angiogenesis, and the ability to invadeand thrive in ectopic tissue environments (see, e.g., Hanahan andWeinberg (2000) Cell 100:57-70).

The mammalian immune response is based on a series of complex cellularinteractions, called the “immune network”. Recent research has providednew insights into the inner workings of this network. While it remainsclear that much of the response does, in fact, revolve around thenetwork-like interactions of lymphocytes, macrophages, granulocytes, andother cells, immunologists now generally hold the opinion that solubleproteins, known as lymphokines, cytokines, or monokines, play a criticalrole in controlling these cellular interactions.

Lymphokines mediate cellular activities in a variety of ways. They havebeen shown to support the proliferation, growth, and differentiation ofpluripotential hematopoietic stem cells into vast numbers of progenitorscomprising diverse cellular lineages making up a complex immune system.Proper and balanced interactions between the cellular components arenecessary for a healthy immune response.

Recently, a novel IL-7-like cytokine cloned from a murine thymic stromalcell line was identified as thymic stromal lymphoietin (TSLP) (see,e.g., J. E. Sims et al., (2000) J. Exp. Med. 192:671-680; U.S. Ser. No.09/963,347, filed Sep. 24, 200; and SEQ ID NOs: 1, and 2). Theactivities of TSLP overlaps with those of IL-7; both stimulatethymocytes and mature T cells and facilitate B lymphopoiesis in culturesof fetal liver and bone marrow lymphocyte precursers. It has also beenfound that the receptor for TSLP is a heterodimer that consist of anIL-7-R-α chain and a common γ-like receptor chain (see, e.g., Reche etal., (2001) J. of Immunol. 167:336-343; Y. Tonozuka et al., (2001)Cytogenet Cell Genet. 93:23-25; A. Pandey et al., (2000) Nat. Immunol.1:59-64; L. S. Park et al., (2000) J. Exp. Med. 192:659-670; and SEQ IDNOs: 3, 4, 5, and 6).

Cancer development and metastasis is a multi-step process that involveslocal neoplasmic growth and invasion followed by dissemination to, andre-establishment at, distant sites. The neoplasm or tumor can consist oftransformed cells and infiltration of stromal cells and cells of theimmune system. The ability for a tumor to metastasize is the majordeterminant of cancer-patient mortality. TGF-β, has been implicated inboth tumor suppression and progression (see e.g., Oft et al., (2002)Nat. Cell Bio. 4:487-494).

Many of the molecular and cellular mechanisms mediating the relationshipbetween metastasis, innate immunity and cancer remain unsolved. TGF-βexpression in tumors facilitates the formation of metastasis. Thepresent invention suggests that TSLP is suppressed by TGF-β.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the expression ofthe immune modulator, TSLP is reduced during tumor progression andaddition of exogenous TSLP causes tumor regression.

The present invention provides a method of modulating a neoplasmcomprising contacting the neoplasm with an effective amount of TSLP oran agonist thereof. The neoplasm is a tumor that is cancerous andepithelial derived that includes a breast tumor, colon tumor, lungtumor, ovarian tumor or a prostate tumor. In certain embodiments, themodulating is inhibition of tumor progression by tumor rejection. Thetumor rejection can be tumor size reduction or loss of metastaticpotential. In further embodiments, the neoplasm contains dendritic cellsand the agonist may be a mutein of TSLP, a small molecule or an agonistantibody.

The present invention further provides a method of treating a subjectsuffering from a neoplasm comprising administering to the subject aneffective amount of TSLP or an agonist thereof. The neoplasm is anepithelial derived cancerous tumor that may be a breast tumor, colontumor, lung tumor, ovarian tumor, or a prostate tumor.

Also provided by the present invention is a method of preventingdevelopment of a neoplasm comprising a administration of an effectiveamount of TSLP or agonist thereof to a subject. The neoplasm is anepithelial derived cancerous tumor that may be a breast tumor, colontumor, lung tumor, ovarian tumor, or a prostate tumor. In certainembodiments, TSLP or an agonist thereof may be administered as a vaccineadjuvant.

The present invention also provides a method of diagnosing a neoplasmcomprising contacting a biological sample from a subject, with an TSLPor TLSPR antibody under conditions suitable for the formation of anantibody:antigen complex and detecting the complex.

DESCRIPTION OF THE DETAILED EMBODIMENTS

I. Definitions

The phrase “TSLP” refers to the nucleic and amino acids of SEQ ID NO: 1and SEQ ID NO: 2. “TSLPR” is composed of two subunits, TSLPR and IL-7α,SEQ ID NOs: 4 and 6. The subunits are encoded by the nucleic acids ofTSLPR and IL-7α, SEQ ID NOs: 3 and 5.

The phrase “effective amount” means an amount sufficient to ameliorate asymptom or sign of the medical condition. Typical mammalian hosts willinclude mice, rats, cats, dogs, and primates, including humans. Aneffective amount for a particular patient may vary depending on factorssuch as the condition being treated, the overall health of the patient,the method route and dose of administration and the severity of sideaffects. When in combination, an effective amount is in ratio to acombination of components and the effect is not limited to individualcomponents alone.

An effective amount of therapeutic will decrease the symptoms typicallyby at least about 10%; usually by at least about 20%; preferably atleast about 30%; or more preferably at least about 50%. The presentinvention provides reagents which will find use in therapeuticapplications as described elsewhere herein, e.g., in the generaldescription for treating disorders associated with the indicationsdescribed above. Berkow (ed.) The Merck Manual of Diagnosis and Therapy,Merck & Co., Rahway, N.J.; Thorn, et al. Harrison's Principles ofInternal Medicine, McGraw-Hill, NY; Gilman, et al. (eds.) (1990) Goodmanand Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed. (1990),Mack Publishing Co., Easton, Pa.; Langer (1990) Science 249:1527-1533;Merck Index, Merck & Co., Rahway, N.J. and Physician's Desk Reference(PDR); Cotran, et al. (eds), supra; and Dale and Federman (eds.) (2000)Scientific American Medicine, Healtheon/WebMD, New York.

The phrase “neoplasm” means an abnormal mass or colony of cells and/orinfiltrating cells produced or attracted by a relatively autonomous newgrowth of tissue. Infiltrating cells can be dendritic cells, T cells andB cells. Most neoplasms arise from the clonal expansion of a single cellthat has undergone neoplastic transformation. The transformation of anormal to a neoplastic cell can be caused by a chemical, physical, orbiological agent (or event) that directly and irreversibly alters thecell genome. Neoplastic cells are characterized by the loss of somespecialized functions and the acquisition of new biological properties,foremost, the property of relatively autonomous (uncontrolled) growth.Neoplastic cells pass on their heritable biological characteristics toprogeny cells.

The past, present, and future predicted biological behavior, or clinicalcourse, of a neoplasm is further classified as benign or malignant, adistinction of great importance in diagnosis, treatment, and prognosis.A malignant neoplasm manifests a greater degree of autonomy, is capableof invasion and metastatic spread, may be resistant to treatment, andmay cause death. A benign neoplasm has a lesser degree of autonomy, isusually not invasive, does not metastasize, and generally produces nogreat harm if treated adequately.

The phrase “inhibition of tumor progression” refers to stopping tumorcell growth, invasion or metastasis depending on the state of the tumor.It should cover all the necessary steps of the evolution of a tumorincluding neovascularization and angiogenesis.

The phrase “metastatic potential or metastasis” refers to the process bywhich cancer spreads from one part of the body to another, the way ittravels from the place at which it first arose as a primary tumor todistant locations in the body.

The extent to which metastasis occurs varies with the individual type oftumor. Melanoma, breast cancer, lung cancer, colon cancer, and prostatecancer are among the types of cancer that are prone to metastasize. Whenmetastasis takes place, the metastases can form at a variety of sites inthe body, with lymph nodes, lungs, liver, brain and bone marrow beingthe more common sites.

The phrase “mutein” or novel mutant proteins includes fragments,derivatives, and analogs of polypeptides. Recombinant DNA technologyknown to those skilled in the art can be used to create novel mutantproteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

The phrase “vaccine adjuvant” in the context of cancer, refers to apeptide used either alone or combined with different cytokines,non-specific stimulators, whole-cell tumors or antigen presenting cellssuch as dendritic cells (see e.g., Kochman et al, (1999) Current MedicalResearch and Opinion, 15:321-326 and Jager et al, (2002) Current Opinionin Immunol., 14:178-182 and Mendelsohn et al; (2001) The Molecular Basisof Cancer, 2^(nd) Edition). The vaccine adjuvant may be administered forthe prevention, amelioration, or treatment of tumors.

II. General

Thymic Stromal Lymphopoietin (TSLP) was originally discovered in themouse and found to play a similar role as its homologue IL-7 insupporting early B and T cell development (see, e.g., J. E. Sims, supra;S. D. Levin et al., supra; and R. J. Ray, et al., supra). TSLP is anovel epithelial cell-derived cytokine, which induces dendritic cell(DC)-mediated CD4⁺ and CD8⁺ T cell responses. TSLP-activated CD 11c⁺ DCspotently activate and expand CD8⁺ T cells, and induces theirdifferentiation into interleukin (IL)-5 and IL-13-producing effectorsexhibiting poor catalytic activity. Additional CD40L triggering ofTSLP-activated DCs induced CD8⁺ T cells with potent cytolytic activity,producing large amounts of interferon (IFN)-γ, while retaining theircapacity to produce IL-5 and IL-13. TSLP has been found to play a roleas an initiator of T cell responses and suggest that CD40L-expressingcells may act in combination with TSLP (see e.g., Gilliet, et al. (2003)J. Exp. Med. 197:1059-1063). The present invention is based on thesurprising results that TSLP is a critical mediator in the developmentof neoplasms.

Recent studies analyzing TSLP suggest that it has potent effects on theability of DCs to induce naïve CD8+ T cell activation anddifferentiation into IL-5 and IL-13 producing T cells (see e.g.,Gilliet, et al. (2003) J. Exp. Med. 197:1059-1063). Activation of DCsappears to be a critical step in the pathogenesis of T cell mediatedresponses to tumors. The molecular mechanism underlying the signaling ofDCs to induce T cell diseases is not clearly understood. The presentfindings that TSLP is highly expressed by a mouse squamous carcinomacell line B9 suggest that TSLP represents a critical factor inunderstanding cancer. Studies in mouse models confirm the role that TSLPplays in tumor suppression.

Mouse squamous carcinoma cell line B9 and a derivative line, RS1, whichcarries overexpressed and activated Smad2 and H-ras, were studied forthe mRNA expression level of the inflammatory cytokine, TSLP. It wasobserved that the B9 cell line expresses high levels of TSLP, whereasthe RS1 cell line expresses strongly reduced levels of TSLP. Furtherstudies were done by passing tumor cells into immune competent mice toobserve the expression of TSLP.

Expression levels of TSLP were measured in various normal and tumorbearing tissues. TSLP expression was significantly higher in normalbreast tissues when compared to proximal breast carcinoma tissues.Similar results were observed with normal colon, lung, ovarian, andprostate cancer tissues and proximal colon, lung, ovarian, and prostatecancer tissues. An interesting observation was that human tumor cellsexpressed significantly diminished amounts of TSLP when compared to theuntransformed counterpart.

TSLP expression in carcinogen treated (DMBA, a site specific carcinogen,and TPA, a tumor promoter) mouse skin were initially elevated at 5 and24 hour timepoints and subsequently fell to at or below control levelsat later timepoints, again demonstrating that progression of theproliferative disorder coincides with a drop in TSLP expression.

It has been shown that TSLP and IL-7 engage similar signal transductionmechanisms. Both induce tyrosine phosphorylation of the transcriptionfactor Stat5. While IL-7 mediated signaling occurs via activation ofJanus kinases Jak1 and Jak 3; TSLP is unable to activate either enzyme,but may instead interact with Jak 2. It was further shown that thefunctional receptor for TSLP, TSLPR, when activated leads tophosphoralation of both Stat5 and Stat3 (see, e.g., Reche, et al. (2001)J. Immunol. 167:336-343).

In an in vivo model of tumor rejection, tumors induced in mice by Ep-rascells were treated by a single administration of adenvirus containingmTSLP (Ad-TSLP) or by several administrations of mTSLP protein. In bothcases, mTSLP appeared to significantly inhibit tumor growth whencompared to control (Ad-GFP or PBS) treatment. This data indicates thatTSLP exhibits potent tumor rejection activity probably caused byreactivation of the immune response to established tumors.

As noted above, inflammation appears to be a critical component of tumorprogression, as many cancers arise in areas of infection, chronicirritation, and inflammation. The tumor microenvironment is anindispensable participant in the neoplastic process, fosteringproliferation, survival and migration. This microenvironment appears tobe largely orchestrated by inflammatory cells. The critical role of TSLPin the inflammatory process and endothelial growth factor activity, withthe data presented above, one skilled in the art would recognize thatthe modulation of TSLP expression and activity is an important componentin the progression of tumors.

The present invention provides methods and reagents to enhance the Th2mediated response by agonizing the activities of TSLP. Enhancement ofthis response is useful in the treatment of disorders due to suppressionof the immune system, e.g., cancer. Augmentation of dendritic cellactivity induced by TSLP will be useful in the treatment of cancer. TSLPand/or agonists thereof will also be useful as a vaccine adjuvant.

II. Agonists and Antagonists

Agonists include the full-length cytokine protein, TSLP (see e.g., SEQID NO: 2). Peptides of those sequences, or variants thereof, will beused to induce receptor signaling. These variants include those that areconservatively substituted and with long half lives. Agonists can bemodified to have longer half-lives by pegalation (PEG) or by tagging theagonist with the Fc portion of an IgG antibody (see e.g., Karmonos,(2001) BioDrugs, 15:705-711). Both methods provide a method to evade theimmune response by not degrading as fast. For example, conjugation ofthe agonist with PEG significantly decreases the proteins clearance fromplasma. Also contemplated are small molecules that also induce receptorsignaling.

Antibodies can be raised to various epitopes of the TSLP proteins,including species, polymorphic, or allelic variants, and fragmentsthereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to TSLPs ineither their active forms or in their inactive forms, including nativeor denatured versions. Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective TSLPs, or screened for agonistic orantagonistic activity, e.g., mediated through a receptor.

A TSLP antibody can also be useful in diagnostic applications. Ascapture or non-neutralizing antibodies, they can be screened for abilityto bind to the antigens without inhibiting binding to a receptor. Asneutralizing antibodies, they can be useful in competitive bindingassays. They will also be useful in detecting or quantifying TSLPprotein or its receptors (see, e.g., Chan (ed. 1987) Immunology: APractical Guide, Academic Press, Orlando, Fla.; Price and Newman (eds.1991) Principles and Practice of Immunoassay, Stockton Press, N.Y.; andNgo (ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y.). Crossabsorptions, depletions, or other means will provide preparations ofdefined selectivity, e.g., unique or shared species specificities. Thesemay be the basis for tests that will identify various groups ofantigens.

Further, the antibodies, including antigen-binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding, e.g., to a receptor that may elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds toantigen, a cell expressing it, e.g., on its surface, is killed. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker, and may effect drugtargeting.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. The antibodies of this invention canalso be useful in diagnostic applications. As capture ornon-neutralizing antibodies, they can be screened for ability to bind tothe antigens without inhibiting binding to a receptor. As neutralizingantibodies, they can be useful in competitive binding assays. They willalso be useful in detecting or quantifying TSLP protein or its receptors(see, e.g., Chan (ed. 1987) Immunology: A Practical Guide, AcademicPress, Orlando, Fla.; Price and Newman (eds. 1991) Principles andPractice of Immunoassay, Stockton Press, N.Y.; and Ngo (ed. 1988)Nonisotopic Immunoassay, Plenum Press, N.Y.). Cross absorptions,depletions, or other means will provide preparations of definedselectivity, e.g., unique or shared species specificities. These may bethe basis for tests that will identify various groups of antigens.

Antibodies raised against each TSLP will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

III. Therapeutic Compositions and Diagnostic Methods

Cytokines, such as IL-7, IL-12 or IL-23 antagonist and agonists thereofare normally administered parentally, preferably intravenously. Sincesuch proteins or peptides may be immunogenic they are preferablyadministered slowly, either by a conventional IV administration set orfrom a subcutaneous depot, e.g. as taught by Tomasi, et al, U.S. Pat.No. 4,732,863. Means to minimize immunological reactions may be applied.Small molecule entities may be orally active.

Parenteral therapeutics may be administered in aqueous vehicles such aswater, saline, or buffered vehicles with or without various additivesand/or diluting agents. Alternatively, a vaccine adjuvant or, asuspension, such as a zinc suspension, can be prepared to include thepeptide. Such a suspension can be useful for subcutaneous (SQ) orintramuscular (IM) injection (see, e.g., Avis, et al. (eds.)(1993)Pharmaceutical Dosage Forms: Parenteral Medications 2d ed., Dekker, NY;Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2ded., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY; Fodor, et al. (1991) Science251:767-773, Coligan (ed.) Current Protocols in Immunology; Hood, et al.Immunology Benjamin/Cummings; Paul (ed.) Fundamental Immunology;Academic Press; Parce, et al. (1989) Science 246:243-247; Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011; and Blundell and Johnson(1976) Protein Crystallography, Academic Press, New York.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells, timing of administration, absorptionthrough epithelial layers, etc. Preferably, an administration regimenmaximizes the amount of therapeutic delivered to the patient consistentwith an acceptable level of side effects. Accordingly, the amount ofbiologic delivered depends in part on the particular entity and theseverity of the condition being treated. Guidance in selectingappropriate doses of cytokine or small molecules is determined usingstandard methodologies.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent.

Antibodies, antibody fragments, and proteins or polypeptides can beprovided by continuous infusion, or by doses at intervals of, e.g., oneday, one week, or 1-7 times per week. Doses may be providedintravenously, subcutaneously, topically, orally, nasally, rectally,intramuscular, intracerebrally, or by inhalation. A preferred doseprotocol is one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects. A total weekly dose is generallyat least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, mostgenerally at least 0.5 μg/kg, typically at least 1 μg/kg, more typicallyat least 10 μg/kg, most typically at least 100 μg/kg, preferably atleast 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably atleast 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at least 25mg/kg, and most optimally at least 50 mg/kg, see, e.g., Yang, et al.(2003) New Engl. J. Med. 349:427-434; Herold, et aL (2002) New Engl. J.Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al. (2003) Cancer Immunol. Immunother.52:133-144. The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

The present invention also provides for administration of biologics incombination with known therapies, e.g., steroids, particularlyglucocorticoids, which alleviate the symptoms, e.g., associated withinflammation, or antibiotics or anti-infectives. Daily dosages forglucocorticoids will range from at least about 1 mg, generally at leastabout 2 mg, and preferably at least about 5 mg per day. Generally, thedosage will be less than about 100 mg, typically less than about 50 mg,preferably less than about 20 mg, and more preferably at least about 10mg per day. In general, the ranges will be from at least about 1 mg toabout 100 mg, preferably from about 2 mg to 50 mg per day. Suitable dosecombinations with antibiotics, anti-infectives, or anti-inflammatoriesare also known.

Antibodies which specifically bind TSLP or TSLPR may be used for thediagnosis of conditions or diseases characterized by expression of TSLPor TSLPR, or in assays to monitor patients being treated with TSLP orTSLPR, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TSLP or TSLPRinclude methods that utilize the antibody and a label to detect TSLP orTSLPR in human body fluids or extracts of cells or tissues. Theantibodies may be used with or without modification, and may be labeledby joining them, either covalently or non-covalently, with a reportermolecule. A wide variety of reporter molecules, which are known in theart, may be used, several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for TSLP or TSLPRare known in the art and provide a basis for diagnosing altered orabnormal levels of TSLP or TSLPR expression. Normal or standard valuesfor TSLP or TSLPR expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to TSLP or TSLPR under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods, but preferably by photometric, means. Quantities ofTSLP or TSLPR expressed in subject, control and disease, samples frombiopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease. The polynucleotides, which may be used, includeoligonucleotide sequences, complementary RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantitate geneexpression in biopsied tissues in which expression of TSLP or TSLPR maybe correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of TSLP orTSLPR, and to monitor regulation of TSLP or TSLPR levels duringtherapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TSLP or TSLPR or closely related molecules, may be used toidentify nucleic acid sequences which encode TSLP or TSLPR. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., 10 unique nucleotides in the 5′ regulatory region, or aless specific region, e.g., especially in the 3′ coding region, and thestringency of the hybridization or amplification (maximal, high,intermediate, or low) will determine whether the probe identifies onlynaturally occurring sequences encoding TSLP or TSLPR, alleles, orrelated sequences. Probes may also be used for the detection of relatedsequences, and should preferably contain at least 50% of the nucleotidesfrom any of the TSLP or TSLPR encoding sequences. The hybridizationprobes of the subject invention may be DNA or RNA and derived from thenucleotide sequence of SEQ ID NO: 1 or from genomic sequence includingpromoter, enhancer elements, and introns of the naturally occurring TSLPor TSLPR.

Means for producing specific hybridization probes for DNAs encoding TSLPor TSLPR include the cloning of nucleic acid sequences TSLP or TSLPR orderivatives thereof into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Antibodies as well as polynucleotide sequences encoding TSLP or TSLPRmay be used for the diagnosis of conditions or disorders which areassociated with expression of TSLP or TSLPR. Examples of such conditionsor disorders include carcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma and particularly cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding TSLP or TSLPR may be used in Southern or northernanalysis, dot blot, or other membrane-based technologies; in PCRtechnologies; or in dipstick, pin, ELISA assays or microarrays utilizingfluids or tissues from patient biopsies to detect TSLP or TSLPRexpression. Such qualitative or quantitative methods are well known inthe art.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES

I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York. Methods forprotein purification include such methods as ammonium sulfateprecipitation, column chromatography, electrophoresis, centrifugation,crystallization, and others (see, e.g., Ausubel, et al. (1987 andperiodic supplements); Deutscher (1990) “Guide to Protein Purification”in Meth. Enzymol., vol. 182, and other volumes in this series; andmanufacturer's literature on use of protein purification products, e.g.,Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif.). Combinationswith recombinant techniques allow fusion to appropriate segments, e.g.,to a FLAG sequence or an equivalent that can be fused via aprotease-removable sequence (see, e.g., Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QIAGEN, Inc., Chatsworth, Calif.).

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

II. TSLP Expression in Human Tumor and Normal Cell Line

Human carcinoma cell lines were chosen that were either part of the NCI60 panel (http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html,A. Monks et al, J. Natl. Cancer Inst. (1991) 83, 757.), or commonly usedin the literature. Corresponding normal cell types from each organ orsystem were obtained from ATCC, Cascade biologics (Portland, Oreg.), orClonetics (Division of Biowhittaker, Walkersville, Md.). In each case,these were normal, non-transformed, non-immortalized cells, andrepresent cell types of the particular anatomic region, e.g.fibroblasts, melanocytes and keratinocytes to serve as normal controlsfor melanoma cell lines, normal mammary epithelial cells to serve asnormal controls for breast carcinoma cell lines, etc.

Cells were grown in tissue culture under standard growth conditions,then harvested. Total RNA was isolated using standard guanidiumisothocyanate/cesium chloride gradients (Sambrook, J., Fritsch E F,Maniatis T: Molecular Cloning: A Laboratory Manual. New York: ColdSpring Harbor Laboratory Press, 1989). Total RNA was subjected totreatment with DNase to eliminate possible genomic DNA contamination.DNase treated total RNA was reverse transcribed using Superscript II(Gibco/BRL) according to manufacturer's instructions. Primers weredesigned using Primer Express (PE Biosystems, Foster City, Calif.).Real-time quantitative PCR on 50 ng of cDNA from each sample wasperformed using Perkin Elmer SYBR green real-time quantitative PCRassay, using an ABI 5700 instrument. Ubiquitin levels were also measuredfor each sample and used to normalize starting quantities of RNA. Thevalues represent the data normalized to ubiquitin.

III. Analysis of Expression of TSLP mRNA in Transformed Mouse TumorCells

The H-Ras transformed mouse keratinocyte cell line PDV and the H-Rastransformed mouse mammary cell line EP2 were treated for 5 days with 5ng/ml TGFbeta1 (R&D systems) or left untreated. mRNA was prepared usingRNeasy columns (Qiagen) (see e.g., Oft et al., (1996) Genes and Dev.10:2462-2477).

B9 squamous carcinoma cells and their derivatives stably overexpressingmutant H-ras and an activated mutant of mSmad2 (see, e.g., Oft et al.(2002) Nature Cell Biol. 4:487-494) were cultured to confludence. mRNAwas isolated using Qiagen RNAeasy RNA isolation kit. Subsequently theRNA was analyzed by real-time PCR using mTSLP RNA specific primers andnormalized to ubiquitin mRNA controls.

IV. TSLP Expression Is Lost During Tumor Passage

The Ras transformed Balb/C mouse mammary epithelial cell line, EP2, wasinjected in nude mice and in syngenic Balb/C mice. Tumor cells werereisolated from the tumor, selected in 0.8 mg/ml G418 (Invitrogen) forfour days. mRNA was isolated as described above (see e.g., Oft et al.,(1996) Genes and Dev. 10:2462-2477).

V. TGFβ Represses TSLP mRNA Expression in Epithelial Tumor Cell Lines

The Ep2 and PDV (DMBA transformed mouse keratinocytes) cell lines werecultured at 60% confluence. Cells were treated with 5 ng/ml TGFβ1 forthree consecutive days. The cells were scraped, spun down, andimmediately snap frozen in liquid nitrogen. TSLP mRNA was isolated andthe expression analyzed by real-time PCR using mTSLP specific RNAprimers. Expression levels were normalized to ubiquitin mRNA controls.

VI. TSLP mRNA Expression and Tumor Progression

Ras transformed epithelial cells were subcutaneiosuly injected insygeneic mice an re-selected after the tumors reached 1 cm³ in volume(see, e.g., Oft et al. (1996) Genes & Devel. 10:2462-2477). All cellwere cultured to 90% confluence. Cells were scraped, spun down, andimmediately snap frozen in liquid nitrogen. mRNA was isolated andsubsequently analyzed by real-time PCR. Expression values werenormalized to ubiquitin mRNA controls.

VII. Tumor Growth and TSLP mRNA Expression

Tumors were induced on immunocompetent Balb/C mice by subcutaneouslyinjecting 1×10⁶ EPXB tumor cells. The experimental group wassubsequently injected with 0.5 mgs of TSLP two times a week followingloss of body weight (see e.g., Oft et al., (1996) Genes and Dev.10:2462-2477).

VIII. TSLP mRNA Expression in Carcinogen Treated Mouse Skin

C57B/6/129 mice were treated topically on the back with 50 ug DMBA or 30ug TPA in 200 ul acetone each, or with acetone alone (control). Smalltissue biopsies were harvested from the same mouse at various timepoints(i.e., 0, 5 h, 24 h, 48 h, and 120 h for DMBA; and 0, 5 h, 24 h, and 72h for TPA). The specimens were immediately smap frozen in liquidnitrogen. Subsequently, mRNA was extracted and analyzed by real-time PCRusing mTSLP RNA specific primers and normalized to upbiquitin mRNAcontrols.

IX. TSLP and Tumor Resistance

Primary NHEK cells (Clonetics, Cambrex) were cultured in definedkeartinocyte growth media (Invitrogen) for five passages. The cells wereinfected at 60% confluency with adenovirus expressing GFP (MOI 100) orleft untreated. The cells were scraped, spun down, and immediately snapfrozen in liquid nitrogen. Subsequently, the RNA was extracted ananalyzed by real time PCR using hTSLP RNA specific primers andnormalized to ubiquitin mRNA controls. TSLP mRNA expression showed asignificant increase in expression in the adenovirus infectedkeratinocytes.

To test if the in vitro data above translated into an in vivo setting,10⁶ Ep-ras cells were injected subcutaneously into syngeneic Balb/Cmice. When tumor size reached 200 mm³, adenovirus expressiong mTSLP orGFP was injected into the tail vein at 10¹⁰ particles/mouse. Separately,mTSLP protein or PBS (control) was also injected (5 μg 3×per week) intothe tail veins at several points from day 1 through day 18. In both,tumor size was measured for 30 days.

X. Listing of Sequence Identifiers

-   -   SEQUENCE ID NO: 1 Human TSLP Nucleic Acid sequence.    -   SEQUENCE ID NO: 2 Human TSLP Amino Acid Sequence    -   SEQUENCE ID NO: 3 Human IL-7Rα Nucleic Acid Sequence    -   SEQUENCE ID NO: 4 Human IL-7Rα Amino Acid Sequence    -   SEQUENCE ID NO: 5 Human TSLPR Nucleic Acid Sequence    -   SEQUENCE ID NO: 6 Human TSLPR Amino Acid Sequence

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1. A method of modulating a neoplasm comprising contacting the neoplasmwith an effective amount of TSLP or an agonist thereof.
 2. The method ofclaim 1, wherein the neoplasm is a tumor.
 3. The method of claim 3,wherein the tumor is a cancerous tumor.
 4. The method of claim 3,wherein the neoplasm is an epithelial derived tumor.
 5. The method ofclaim 4, wherein the epithelial derived tumor is a: a) breast tumor; b)colon tumor; c) lung tumor; d) ovary tumor; or e) prostate tumor.
 6. Themethod of claim 1, wherein the modulating is inhibition of tumorprogression.
 7. The method of claim 6, wherein the inhibition of tumorprogression is tumor rejection.
 8. The method of claim 7, wherein thetumor rejection consists of: a) tumor size reduction; or b) loss ofmetastatic potential.
 9. The method of claim 1, wherein the neoplasmcontains dendritic cells.
 10. The method of claim 1, wherein the agonistis a: a) mutein of TSLP; b) small molecule; or c) agonist antibody. 11.A method of treating a subject suffering from a neoplasm comprisingadministering to the subject an effective amount of TSLP or an agonistthereof.
 12. The method of claim 11, wherein the neoplasm is a tumor.13. The method of claim 12, wherein the tumor is a cancerous tumor. 14.The method of claim 13, wherein the neoplasm is an epithelial derivedtumor.
 15. The method of claim 14, wherein the epithelial tumor is a: a)breast tumor; b) colon tumor; c) lung tumor; d) ovary tumor; or e)prostate tumor.
 16. A method of preventing development of a neoplasmcomprising a administration to a subject of an effective amount of TSLPor agonist thereof.
 17. The method of claim 16, wherein the neoplasm isa tumor.
 18. The method of claim 17, wherein the tumor is a canceroustumor.
 19. The method of claim 18, wherein the neoplasm is an epithelialderived tumor.
 20. The method of claim 19, wherein the epithelial tumoris a: a) breast tumor; b) colon tumor; c) lung tumor; d) ovary tumor; ore) prostate tumor.
 21. The method of claim 16, wherein the TSLP oragonist thereof is administered as a vaccine adjuvant.
 22. A method ofdiagnosing a neoplasm comprising contacting a biological sample from asubject with an TSLP or TLSPR antibody under conditions suitable for theformation of an antibody:antigen complex and detecting the complex.